Approximately 40 human chemokines have been described, that function, at least in part, by modulating a complex and overlapping set of biological activities important for the movement of lymphoid cells and extravasation and tissue infiltration of leukocytes in response to inciting agents (See, for example: P. Ponath, Exp. Opin. Invest. Drugs 7:1–18 (1998); Baggiolini, M., Nature 392:565–568 (1998); Locati, et al., Annu. Rev. Med. 50:425–40 (1999)). These chemotactic cytokines, or chemokines, constitute a family of proteins, approximately 8–10 kDa in size. Chemokines appear to share a common structural motif, that consists of 4 conserved cysteines involved in maintaining tertiary structure. There are two major subfamilies of chemokines: the “CC” or β-chemokines and the “CXC” or α-chemokines. The receptors of these chemokines are classified based upon the chemokine that constitutes the receptor's natural ligand. Receptors of the β-chemokines are designated “CCR” while those of the α-chemokines are designated “CXCR.”
Chemokines are considered to be principal mediators in the initiation and maintenance of inflammation (see Chemokines in Disease published by Humana Press (1999), Edited by C. Herbert; Murdoch, et al., Blood 95:3032–3043 (2000)). More specifically, chemokines have been found to play an important role in the regulation of endothelial cell function, including proliferation, migration and differentiation during angiogenesis and re-endothelialization after injury (Gupta, et al., J. Biol. Chem. 7:4282–4287 (1998); Volin, et al., Biochem. Biophys Res. Commun. 242:46–53 (1998)). Two specific chemokines have been implicated in the etiology of infection by human immunodeficiency virus (HIV).
In most instances, HIV initially binds via its gp120 envelope protein to the CD4 receptor of the target cell. A conformational change appears to take place in gp120 which results in its subsequent binding to a chemokine receptor, such as CCR5 (Wyatt, et al., Science 280:1884–1888 (1998); Rizzuto, et al., Science 280:1949–1953 (1998); Berger, et al., Annu. Rev. Immunol. 17:657–700 (1999)). HIV-1 isolates arising subsequently in the infection bind to the CXCR4 chemokine receptor.
Following the initial binding by HIV to CD4, virus-cell fusion results, which is mediated by members of the chemokine receptor family, with different members serving as fusion cofactors for macrophage-tropic (M-tropic) and T cell line-tropic (T-tropic) isolates of HIV-1 (Carroll, et al., Science 276:273–276 (1997); Feng et al., Science 272:872–877 (1996); Bleul, et al., Nature 382:829–833 (1996); Oberlin, et al., Nature 382:833–835 (1996); Cocchi, et al., Science 270:1811–1815 (1995); Dragic, et al., Nature 381:667–673 (1996); Deng, et al., Nature 381:661–666 (1996); Alkhatib, et al., Science 272:1955–1958, (1996)). During the course of infection within a patient, it appears that a majority of HIV particles shift from the M-tropic to the more pathogenic T-tropic viral phenotype (Blaak, et al., Proc. Natl. Acad. Sci. 97:1269–1274 (2000); Miedema, et al., Immune. Rev. 140:35 (1994); Simmonds, et al., J. Virol. 70:8355–8360 (1996); Tersmette, et al., J. Virol. 62:2026–2032, (1988); Connor, R. I., Ho, D. D., J. Virol. 68:4400–4408 (1994); Schuitemaker, et al., J. Virol. 66:1354–1360 (1992)). The M-tropic viral phenotype correlates with the virus's ability to enter the cell following binding of the CCR5 receptor, while the T-tropic viral phenotype correlates with viral entry into the cell following binding and membrane fusion with the CXCR4 receptor. Clinical observations suggest that patients who possess genetic mutations in CCR5 appear resistant, or less susceptible to HIV infection (Liu, et al., Cell 86:367–377 (1996); Samson, et al., Nature 382:722–725 (1996); Michael, et al., Nature Med. 3:338–340 (1997); Michael, et al., J. Virol. 72:6040–6047 (1998); Obrien, et al., Lancet 349:1219 (1997); Zhang, et al., AIDS Res. Hum. Retroviruses 13:1357–1366 (1997); Rana, et al., J. Virol. 71:3219–3227 (1997); Theodorou, et al., Lancet 349:1219–1220 (1997). Despite the number of chemokine receptors which have been reported to HIV mediate entry into cells, CCR5 and CXCR4 appear to be the only physiologically relevant coreceptors used by a wide variety of primary clinical HIV-1 strains (Zhang, et al., J. Virol. 72:9307–9312 (1998); Zhang, et al., J. Virol. 73:3443–3448 (1999); Simmonds, et al., J. Virol. 72:8453–8457 (1988)). Fusion and entry of T-tropic viruses that use CXCR4 are inhibited by the natural CXC-chemokine stromal cell-derived factor-1, whereas fusion and entry of M-tropic viruses that use CCR5 are inhibited by the natural CC-chemokines namely, Regulated on Activation Normal T-cell Expressed and Secreted (RANTES) and Macrophage Inflammatory proteins (MIP-1 alpha and beta).
In addition to serving as a co-factor for HIV entry, the direct interaction of virus-associated gp120 with CXCR4 has been recently suggested as a possible cause of CD8+ T-cell apoptosis and AIDS-related dementia via induction of neuronal cell apoptosis (Hesselgesser, et al., Curr. Biol. 8:595–598 (1998); Hesselgesser, et al., Curr. Biol. 7:112–121 (1997); Hesselgesser, et al., “Chemokines and Chemokine receptors in the Brain” in Chemokines in Disease published by Humana Press (1999), Edited by C. Herbert; Herbein, et al., Nature 395:189–194 (1998); Buttini, et al., Nature Med. 4:441–446 (1998); Ohagen, et al., J. Virol. 73:897–906 (1999); Biard-Piechaczyk, et al., Virology 268:329–344 (2000); Sanders, et al., J. Neuroscience Res. 59:671–679 (2000); Bajetto, et al., J. Neurochem. 73:2348–2357 (1999); Zheng, et al., J. Virol. 73:8256–8267 (1999)).
However, the binding of chemokine receptors to their natural ligands appears to serve a more evolutionary and central role than only as mediators of HIV infection. The binding of the natural ligand, pre-B-cell growth-stimulating factor/stromal cell derived factor (PBSF/SDF-1) to the CXCR4 chemokine receptor provides an important signaling mechanism: CXCR4 or SDF-1 knock-out mice exhibit cerebellar, cardiac and gastrointestinal tract abnormalities and die in utero (Zou, et al., Nature 393:591–594 (1998); Tachibana, et al., Nature 393:591–594 (1998); Nagasawa, et al., Nature 382:635–638 (1996)). CXCR4-deficient mice also display hematopoietic defects (Nagasawa, et al., Nature 382:635–638 (1996)); the migration of CXCR4 expressing leukocytes and hematopoietic progenitors to SDF-1 appears to be important for maintaining B-cell lineage and localization of CD34+ progenitor cells in bone marrow (Bleul, et al., J. Exp. Med. 187:753–762 (1998); Viardot, et al., Ann. Hematol. 77:195–197 (1998); Auiti, et al., J. Exp. Med. 185:111–120 (1997); Peled, et al., Science 283:845–848 (1999); Qing, et al., Immunity 10:463–471 (1999); Lataillade, et al., Blood 95:756–768 (1999); Ishii, et al., J. Immunol. 163:3612–3620 (1999); Maekawa, et al., Internal Medicine 39:90–100 (2000); Fedyk, et al., J. Leukocyte Biol. 66:667–673 (1999); Peled, et al., Blood 95:3289–3296 (2000)).
The signal provided by SDF-1 on binding to CXCR4 may also play an important role in tumor cell proliferation and regulation of angiogenesis associated with tumor growth (See “Chemokines and Cancer” published by Humana Press (1999); Edited by B. J. Rollins; Arenburg, et al., J. Leukocyte Biol. 62:554–562 (1997); Moore, et al., J. Invest. Med. 46:113–120 (1998); Moore, et al., Trends cardiovasc. Med. 8:51–58 (1998); Seghal, et,al., J. Surg. Oncol. 69:99–104 (1998)); the known angiogenic growth factors VEG-F and bFGF, up-regulate levels of CXCR4 in endothelial cells, and SDF-1 can induce neovascularization in vivo (Salcedo, et al., Am. J. Pathol. 154:1125–1135 (1999)); Leukemia cells that express CXCR4 migrate and adhere to lymph nodes and bone marrow stromal cells that express SDF-1 (Burger, et al., Blood 94:3658–3667 (1999); Arai, et al., Eur. J. Haematol. 64:323–332 (2000); Bradstock, et al., Leukemia 14:882–888 (2000); Burger et al., Blood 96:265502663 (2000); Mörle, R. et al. Brit. J. Haematol. 110:561–582 (2000).
The binding of SDF-1 to CXCR4 has also been implicated in the pathogenesis of atherosclerosis (Abi-Younes, et al., Circ. Res. 86:131–138 (2000)), renal allograft rejection (Eitner, et al., Transplantation 66:1551–1557 (1998)), asthma and allergic airway inflammation (Yssel, et al., Clinical and Experimental Allergy 28:104–109 (1998); Nagase, et al., J. Immunol. 164:5935–5943 (2000); Gonzalo, et al., J. Immunol. 165:499–508 (2000)), Alzheimer's disease (Xia, et al., J. Neurovirology 5:32–41 (1999)) and arthritis (Nanki, et al., J. Immunol. 164:5010–5014 (2000)).
In attempting to better understand the relationship between chemokines and their receptors, recent experiments to block the fusion, entry and replication of HIV via the CXCR4 chemokine receptor were carried out through the use of monoclonal antibodies or small molecules that appear to suggest a useful therapeutic strategy (Schols, et al., J. Exp. Med. 186:1383–1388 (1997); Schols, et al., Antiviral Research 35:147–156 (1997); Bridger, et al., J. Med. Chem. 42:3971–3981 (1999); Bridger, et al., “Bicyclam Derivatives as HIV Inhibitors” in Advances in Antiviral Drug Design Volume 3, p161–229; Published by JAI press (1999); Edited by E. De Clercq). Small molecules, such as bicyclams, appear to specifically bind to CXCR4 and not CCR5 (Donzella, et al., Nature Medicine 4:72–77 (1998)). These experiments demonstrated interference with HIV entry and membrane fusion into the target cell in vitro. More recently, bicyclams were also shown to inhibit fusion and replication of Feline Immunodeficiency Virus (FIV) that uses CXCR4 for entry (Egberink, et al., J. Virol. 73:6346–6352 (1999)).
Additional experiments have shown that the bicyclam dose-dependently inhibits binding of 125I-labeled SDF-1 to CXCR4 and the signal transduction (indicated by an increase in intracellular calcium) in response to SDF-1. Thus, the bicyclam also functioned as an antagonist to the signal transduction resulting from the binding of stromal derived factor or SDF-1α, the natural chemokine to CXCR4. Bicyclams also inhibited HIV gp120 (envelope)-induced apoptosis in non-HIV infected cells (Blanco, et al., Antimicrobial Agents and Chemother. 44:51–56 (2000)).
U.S. Pat. No. 5,583,131, U.S. Pat. No. 5,698,546, U.S. Pat. No. 5,817,807, U.S. Pat. No. 5,021,409 and U.S. Pat. No. 6,001,826, which are incorporated herein by reference, disclose cyclic compounds that are active against HIV-1 and HIV-2 in in vitro tests. It was subsequently disclosed, in published PCT application PCT/CA99/00619 (WO 00/029870), incorporated herein by reference, that these compounds as well as additional compounds with less complex structures exhibit anti-HIV activity by binding to the chemokine receptor CXCR4 and/or CCR5 expressed on the surface of certain cells of the immune system. This competitive binding protects these target cells, which utilize the CXCR4 receptor for entry from infection by HIV. In addition, the compounds antagonize the binding, signaling and chemotactic effects of the natural ligand for CXCR4, i.e., the chemokine stromal cell-derived factor 1α (SDF-1), and also have protective effects against HIV infection of target cells by binding in vitro to the CCR5 receptor.
Additionally, published PCT application PCT/CA00/00104, (WO 00/45814), incorporated herein by reference, discloses that the cyclic polyamine antiviral agents described in the above-mentioned documents have the effect of enhancing production of white blood cells as well as exhibiting antiviral properties. Thus, these agents are useful for controlling the side-effects of chemotherapy, enhancing the success of bone marrow transplantation, enhancing wound healing and bum treatment, as well as combating bacterial infections in leukemia.
Published PCT application PCT/CA00/00321 (WO 00/56729), incorporated herein by reference, discloses additional heterocyclic compounds that exhibit anti-HIV activity by binding to the chemokine receptors CXCR4 and CCR5 expressed on the surface of certain cells of the immune system. This competitive binding thereby protects these target cells, which utilize the CXCR4 or CCR5 receptors for entry, from infection by HIV. In addition, these compounds antagonize the binding, signaling and chemotactic effects of the natural ligand for CXCR4, i.e., the chemokine stromal cell-derived factor 1α (SDF-1) and/or the natural ligand for CCR5, the chemokine RANTES.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. Further, all documents referred to throughout this application are hereby incorporated in their entirety by reference herein.
The present invention describes novel compounds that exhibit protective effects against HIV infection of target cells by binding to the chemokine receptors CXCR4 or CCR5, in a similar manner to the previously disclosed macrocyclic compounds, and that are additionally useful in other indications addressed by the compounds as described above.